Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer–Villiger Monooxygenase

The typically low thermodynamic and kinetic stability of enzymes is a bottleneck for their application in industrial synthesis. Baeyer–Villiger monooxygenases, which oxidize ketones to lactones using aerial oxygen, among other activities, suffer particularly from these instabilities. Previous efforts in protein engineering have increased thermodynamic stability but at the price of decreased activity. Here, we solved this trade-off by introducing mutations in a cyclohexanone monooxygenase from Acinetobacter sp., guided by a combination of rational and structure-guided consensus approaches. We developed variants with improved activity (1.5- to 2.5-fold) and increased thermodynamic (+5 °C Tm) and kinetic stability (8-fold). Our analysis revealed a crucial position in the cofactor binding domain, responsible for an 11-fold increase in affinity to the flavin cofactor, and explained using MD simulations. This gain in affinity was compatible with other mutations. While our study focused on a particular model enzyme, previous studies indicate that these findings are plausibly applicable to other BVMOs, and possibly to other flavin-dependent monooxygenases. These new design principles can inform the development of industrially robust, flavin-dependent biocatalysts for various oxidations.


Rossmann fold sequence in BVMOs
The Rossmann fold is a super secondary structure seen in proteins binding nucleotides. It is composed of beta sheets and alpha-helical sections. The alpha helices surround both faces of the sheet to produce a three-layered sandwich, and the beta-strands are hydrogen-bonded to each other forming an extended beta-sheet. The main function of this motif in enzymes is the binding of nucleotide cofactors and/or substrates. The first, third, and sixth position in the Rossman fold is highly conserved and susceptible to changes. In CHMO Acineto , the Rossmann fold sequence is composed of -GGGFGGand is crucial for FAD-binding ( Figure S1). This motif is also highly conserved within the BVMO family. Interestingly in more thermostable variants, a glycine substitution into an alanine is observed. This is especially true for TmCHMO and PAMO ( Figure S2).

A III Prediction of mutants by structure-guided consensus approach
With the assumptions that primordial Earth was hotter than today's climate and thus functional proteins had, on average, to be more stable and that encoding stability in the amino acid sequence follows a Boltzmann-like or similar statistics, the consensus amino acid at each position contributes to an overall stable protein. In cases of low level of sequence identity and/or few available amino acid sequences, however, the consensus residue of a specific position cannot be found easily or not at all. The structure-guided consensus approach utilizes structural information in addition to the frequency of an amino acid in a specific position of the sequence to point out residues for stabilization but then to reduce the number of residues in a given protein to be mutated and checked for stabilization. [2][3] A IV

Homology model of all three generations of CHMO A c i n e t o
The structures were visualized by PyMOL 4 using first-ever crystalized CHMO Acineto 5 , which is a mutated variant as the template. The mutations introduced in this study are colored in red. The FAD is in yellow color. Figure S3. A) first generation B) second generation C) third generation variants locations on a homology model of CHMO Acineto . Homology models were made based on the crystal structure of the CHMO Acineto mutant 5 as the template. Pymol was used to visualize the structures. 4 A V

Evaluation of melting temperature (T m )
Data analysis has been performed using NT Melting Control software (NanoTemper Technologies GmbH). The melting temperature (T m ) was determined by fitting the tryptophan fluorescence emission ratio of 350 nm to 330 nm using a polynomial function, in which the maximum slope is indicated by the peak of its first derivative.
A VII

Characterization of site saturated G14 position
Site saturation mutagenesis was carried out by using the Q5 site directed mutagenesis kit. This was performed to evaluate the effect of different amino acids in position 14. The primers were designed using the NEBbaseChanger (https://nebasechanger.neb.com). n.a n.a n.a G14D 23 n.a 30 G14P n.a n.a n.a G14K n.a n.a n.a n.a= not applicable A VII.1

Circular dichroism (CD) spectroscopy
CD spectroscopy is a useful technique to investigate the secondary structure and folding properties of proteins 9 . This technique is helping to determine if the purified protein is folded, or if a mutation affects its conformation. As it is shown in Figure S5, the only mutation that affected enzyme structure is the substitution of glycine with cysteine (G14C). This means that the reason why the variants are not active is not due to unfolding. The variants are either fragile, and thus can't not survive the purification process and lose their activity, or have no ability anymore to bind to FAD to perform the reaction. A VIII

Substrate profile
The substrate scope and the enantioselectivity of a few variants were evaluated to determine if the mutations have changed the selectivity of variants or not. The reactions were performed overnight at 30℃ in a shaking incubator operating at 200 rpm (Table S6 & Figure S7).  Figure S6. List of compounds used for the enantioselectivity evaluation.

A IX MD simulation and structure analysis
The protonation states of the amino acids were assigned based on a pH 7 and oxidized form of FAD to reproduce the resting state of the enzyme. All the MD simulations were performed using the GROMOS simulation package (https:/www.gromos.net) 10 , and the models were parameterized using the 54A8 GROMOS forcefield for both the protein and the FAD 11 . Short energy minimizations were performed using the steepest-descent algorithm in the vacuum. The models were then placed in a periodic rectangular water box with simple point charge (SPC) water molecules, leaving a minimum distance of 1.4 nm from the solute to the box walls. The systems were further minimized using the steepest-descent algorithm with position restraints on the solute atoms. Counter ions were added by replacing water molecules with the most favorable electrostatic potential to reach charge neutralization using the program ion provided in the GROMOS++ package. 12 Five replicas for each model were generated by setting different initial velocities sampled from a Maxwell-Boltzman distribution at 60 K.
The systems were thermalized up to 300 K by five discrete steps with position restraints on the solute atoms. The strength of the restraints was decreased by a factor 10 from 2.5 × 104 to 0 kJ mol−1nm−2.
Production simulations of 50 ns each were performed at a constant temperature of 300 K and a constant pressure of 1 atm using the Nosé-Hoover-chains algorithm for the temperature control with

B I Plasmid map and cloning plan
All the variants in this study were cloned in pET22 b (+) by using the NdeI, and NotI restriction sites, and all contained the His-Tag at C-terminal. The first and second series of mutants have been prepared by Dr. Saima Feroz 18

B II Site saturation mutagenesis
Site saturation mutagenesis of G14 residue was carried out using the Q5 site directed mutagenesis kit according to the protocol provided by the kit.
The mutations within the library (1 st -3 rd generation) were introduced using the QuikChange® II XL Site-

B VII Melting temperature (T m )
The melting temperature (T m ) was measured by Prometheus NT.48. The samples were prepared in TrisHCl 50 mM pH 8.5, 10 µM FAD with a final enzyme concentration of 2 mg mL -1 . The glass capillaries were filled with 10 µL of the enzyme solution, and the samples ran from 20 to 95 °C. Data were analysed using NT Melting Control software (NanoTemper Technologies GmbH). The melting temperature (Tm) was determined by fitting the tryptophan fluorescence emission ratio of 350 nm to 330 nm using a polynomial function, in which the maximum slope is indicated by the peak of its first derivative.

Biotransformation
The resting cell method was used to perform the biotransformation. Recombinant protein expression was done as described before. Cells were collected by centrifugation (6000 rpm, 4 °C, 10 min), resuspended and washed in 50 mM PBS buffer pH 7.4. After washing, the cells were centrifuged (6000 rpm, 4 °C, 10 min) and resuspended again with the same buffer to reach OD 590 = 30. The reaction contained 1 mL (OD 590 =30) of recombinant expressed cells suspended in PBS buffer (pH 7.4, 50 mM) and 10 mM substrate final concentration (methanol as cosolvent (5 % of total volume)).
The components of the reaction (1.02 mL in total) were added into 25 mL flask, and the reaction was performed at 30 °C by shaking (220 rpm) for 24 h. 19 The product was extracted with ethyl acetate containing 0.1 mM methyl benzoate as the internal standard for the GC analysis. The product analysis was performed with GC (Thermo Scientific Trace or Focus GC, Thermo Fisher Scientific, Waltham, MA, USA) using the chiral/achiral column. Product validation was carried out by literature-known reference biotransformations.